Eps8抗原改造表位 HLA-A*0201限制性抗肿瘤能力观察

摘要

目的：观察人表皮生长因子受体通路底物8（Eps8）抗原改造表位是否有人白细胞抗原A（HLA-A）*0201限制性抗肿瘤能力。方法替换 Eps8抗原锚定位点氨基酸获得改造肽，用 BIMAS、SYFPEITHI 在线数据库及 Aotodock 4．2软件预测改造表位与 HLA-A*0201分子的结合力，筛选出稳定结合的改造表位 E1-9V（ILDDIEF-FV）、E1-1Y9V（YLDDIEFFV）。用经典肽结合力实验检测各改造表位与 HLA-A*0201分子的亲和力。制备天然表位（E1）、E1-9V、E1-1Y9V 特异性杀伤性 T 淋巴细胞（CTLs）。用乳酸脱氢酶（LDH）释放试验检测并比较 E1-9V、E1-1Y9V 特异性 CTLs 对 MCF-7细胞、沉默 Eps8的 MCF-7细胞（MCF-7／shRNA）和抗 HLA-A2抗体预孵育的 MCF-7细胞（MCF-7／A2Ab）的杀伤率，E1-9V、E1-1Y9V 特异性 CTLs 对 SW480、U251、K562、IM-9细胞的杀伤率，E1、E1-9V、E1-1Y9V 特异性 CTLs 对 T2细胞的杀伤率。结果 E1-9V、E1-1Y9V 均可与 HLA-A*0201分子 B、F 对接口袋的氨基酸形成稳定氢键。肽结合力实验结果显示 E1-9V、E1-1Y9V 与 HLA-A*0201均具有高亲和力，且高于天然表位 E1，P 均＜0．05。E1-9V、E1-1Y9V 特异性 CTLs 对 MCF-7／shRNA、MCF-7／A2Ab 细胞杀伤率明显低于 MCF-7细胞，P 均＜0．05。E1-9V、E1-1Y9V 特异性 CTLs 对 SW480、U251细胞杀伤率明显高于 K562、IM-9细胞（P 均＜0．05）。E1-1Y9V 特异性 CTLs 对 T2细胞的杀伤率显著高于 E1、E1-9V 特异性 CTLs，P 均＜0．05。结论 Eps8抗原改造表位 E1-9V、E1-1Y9V 与天然表位 E1相比具有更高的 HLA-A*0201分子亲和力，保留了原有的免疫原性，并且 E1-1Y9V 抗肿瘤免疫效应强于天然表位 E1。%Objective To observe whether modified epitopes from the human epidermal growth factor receptor pathway substrate 8 (Eps8)antigen have leucocyte antigen (HLA -A)*0201 restrictive anti-tumor ability.Methods We obtained the modified peptides from Eps8 containing HLA-A*0201-binding anchor motifs by replacing anchor residues,then we se-lected stable binding affinity peptides E1-9V (ILDDIEFFV)and E1-1Y9V(YLDDIEFFV)by using different computer al-gorithms such as BIMAS,SYFPEITHI database and Aotodock 4.2 software to predict the modified peptides and their bind-ing affinity to HLA-A*0201,respectively.T2 binding assay were performed to verify modified epitopes'binding affinity to HLA-A*0201.Cytotoxic T lymphocytes (CTLs)were induced from peripheral blood mononuclear cells (PBMCs)of HLA-A*0201 positive healthy donors stimulated by modified epitopes E1-9V and E1-1Y9V.The kill rates on SW480, U251,K562,IM-9 and T2 cells were compared between E1-9V and E1-1Y9V peptide-specific CTLs,and meanwhile,the kill rates on T2 cells were compared among E1,E1-9V and E1-1Y9VE1-9V peptide-specific CTLs.Results Both E1-9V and E1-1Y9V formed stable hydrogen bonds with amino acids of HLA-A*0201 molecular B,F docking pockets.T2 binding assay showed that E1-9V and E1-1Y9V showed significantly higher affinity for HLA-A*0201 than the native peptide E1 (all P <0.05).Modified peptide-specific CTLs of E1-9V and E1-1Y9V had lower kill rates on MCF-7 /shRNA and MCF-7 /A2Ab cells than that of MCF-7 cells (all P <0.05).In addition ,the CTLs expressed higher cytotoxicity against tumor cells SW480 and U251 than that of K562 and IM-9 (all P <0.05).E1-1Y9V peptide-specific CTLs showed higher cytotox-icity against tumor cells T2 than E1 and E1-9V peptide-specific CTLs (all P <0.05).Conclusion Compared with native peptide E1,modified epitopes E1-9V and E1-1Y9V have higher binding affinity with HLA-A*0201 and retain immunoge-necity.In addition,the anti-tumor immunity effect of modified epitope E1-1Y9V is stronger than native peptide E1.